Pmxs ires blasticidin retroviral vector

Full-length COL7A1^c.425A>G was cloned by the Golden Gate cloning technique using NEBuilder^® Hifi DNA Assembly Cloning. The whole 9.2 kB cDNA was split into two fragments (F1: 4461 bp; F2: 4440 bp) and cloned into a pMXs-IRES-Blasticidin Retroviral vector (Cell Biolabs, San Diego, CA, USA) (F3: 5636 bp). Primers were designed using the NEBridge ^® Golden Gate Assembly Tool. Amplification of the fragments was performed using Phusion^TM High-Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA) for F1 at an annealing temperature of 63.4 °C (forward primer: 5′-cctcgagggccggcgcgccgcATGACGCTGCGGCTTCTG-3′; reverse primer: 5′-cctttggggccAATAGCTCCAGGAGGTCCC-3′) and for F2 at an annealing temperature of 66.2 °C (forward primer: 5′-ctggagctattGGCCCCAAAGGTGACCGG-3′; reverse primer: 5′-gaggggcggaatttacgtagcTCAGTCCTGGGCAGTACCTG-3′). The vector was linearized with NotI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and cloned according to the NEBuilder^® Hifi DNA Assembly Cloning (New England Biolabs, Ipswich, MA, USA) instructions.